The overall goal of our research proposal is to identify and characterize the molecular signals and the mechanical stimuli that mediate skeletal tissue regeneration. We have developed a mouse model of distraction osteogenesis, a clinical procedure which uses a defined mechanical force to induce the formation of bone. Preliminary data support our hypothesis that the distraction site represents a continually regenerating system where multipotent cells are recruited and differentiate along an osteogenic pathway in a spatially organized manner. Osteoblast differentiation occurs over a protracted period of time, allowing us to study crucial even in this developmental process. In this procedure, forces are transmitted to the bone through an external frame. Thus we have the ability to control mechanical boundary conditions, while simultaneously monitoring the tissue response. Collectively, these attributes make distraction osteogenesis is an ideal model to study the molecular basis of skeletal regeneration and the influence of the mechanical environment on this process. We are taking a multidisciplinary approach investigating the molecular basis of skeletal tissue regeneration and remodeling. In aims 1 and 2, we propose experiments that focus on the origins, identities and the commitment state of cells contributing to the new bone. We will use in situ hybridization and immunolocalization to determine whether the bone marrow and the periosteum contribute osteoprogenitor cells to the distraction gap. We will create a molecular map of the distraction site during the different phases of treatment. Our goal is to use this information 1b develop pharmacological approaches to stimulating osteoprogenitor and to accelerate mineralization of the collagen scaffold. In specific aim 3, we propose experiments to determine the mechanical properties of the regenerated tissue using atomic force microscopy in order to provide a clinically relevant measure of its structural integrity. We will use x-ray tomography to obtain high resolution, three-dimensional images of tie mineralization patterns during different phases of distraction; because this procedure is non- invasive, we can directly compare gene expression patterns from Aims 1 and 2 with mineralization maps. In specific aim 4 we propose to use finite element analysis to define the mechanical environment of the distraction site. The model geometries will be based on histological and molecular data from Aims 1 and 2, and the model properties will be defined by in vitro mechanical testing and by mechanical properties determined in Aim 3. These data will aid us in determining the mechanical environments which are most conducive to the formation and mineralization of determining skeletal tissue. Collectively, these results on the biomimetics of bone regeneration will provide a sound scientific basis for the treatment of musculoskeletal diseases and injuries.